Activation of OX1 orexin receptors heterologously expressed in Chinese hamster ovary (CHO) cells led to a rapid, strong, and long-lasting increase in ERK phosphorylation (activation). Dissection of the signal pathways to ERK using multiple inhibitors and dominant-negative constructs indicated involvement of Ras, protein kinase C, phosphoinositide-3-kinase, and Src. Most interestingly, Ca2+ influx appeared central for the ERK response in CHO cells, and the same was indicated in recombinant neuro-2a cells and cultured rat striatal neurons. Detailed investigations in CHO cells showed that inhibition of the receptor- and store-operated Ca2+ influx pathways could fully attenuate the response, whereas inhibition of the store-operated Ca2+ influx pathway alone or the Ca2+ release was ineffective. If the receptor-operated pathway was blocked, an exogenously activated store-operated pathway could take its place and restore the coupling of OX1 receptors to ERK. Further experiments suggested that Ca2+ influx, as such, may not be required for ERK phosphorylation, but that Ca2+, elevated via influx, acts as a switch enabling OX1 receptors to couple to cascades leading to ERK phosphorylation, cAMP elevation, and phospholipase C activation. In conclusion, the data suggest that the primary coupling of orexin receptors to Ca2+ influx allows them to couple to other signal pathways; in the absence of coupling to Ca2+ influx, orexin receptors can act as signal integrators by taking advantage of other Ca2+ influx pathways.

Background and purpose: Orexin (OX) receptors induce Ca 2 þ elevations via both receptor-operated Ca 2 þ channels (ROCs) and the "conventional" phospholipase C (PLC)-Ca 2 þ release-store-operated Ca 2 þ channel (SOC) pathways. In this study we assessed the ability of these different Ca 2 þ influx pathways to amplify OX 1 receptor signalling to PLC in response to stimulation with the physiological ligand orexin-A. Experimental approach: PLC activity was assessed in CHO cells stably expressing human OX 1 receptors. Key results: Inhibition of total Ca 2 þ influx by reduction of the extracellular [Ca 2 þ ] to 1 mM effectively inhibited the receptor-stimulated PLC activity at low orexin-A concentrations (by 93% at 1 nM), and this effect was gradually reduced by higher orexin-A concentrations. A similar but weaker inhibitory effect (84% at 1 nM) was obtained on depolarization to B0 mV, which disrupts most of the driving force for Ca 2 þ entry. The inhibitor of the OX 1 receptor-activated ROCs, tetraethylammonium chloride (TEA), was somewhat less effective than the reduction in extracellular [Ca 2 þ ] at inhibiting PLC activation, probably because it only partially blocks ROCs. The partial inhibitor of both ROCs and SOCs, Mg 2 þ , and the SOC inhibitors, dextromethorphan, propoxy)-4-methoxyphenethyl]-1H-imidazole HCl) and 2-APB (2-aminoethoxydiphenyl borate), inhibited PLC activity at low concentrations of orexin-A, but were not as effective as TEA. Conclusions and implications: Both ROCs and SOCs markedly amplify the OX 1 receptor-induced PLC response, but ROCs are more central for this response. These data indicate the crucial role of ROCs in orexin receptor signalling.

We investigated coupling of OX(1) receptors to phospholipase activation and diacylglycerol generation in Chinese hamster ovary (CHO) cells using both biochemical and fluorescence "real-time" methods. The results indicate that at lowest orexin-A concentrations (highest potency), diacylglycerol generated results from phospholipase D activity. At 10-100-fold higher orexin-A concentrations, phospholipase C is activated, likely hydrolyzing phosphatidylinositol (PI) or phosphatidylinositol monophosphate (PIP) but not phosphatidylinositol bisphosphate (PIP(2)). At further 7-fold higher orexin-A concentrations, PIP(2) is hydrolyzed, releasing both diacylglycerol and inositol-1,4,5-trisphosphate. Thus, OX(1) orexin receptors connect to multiple phospholipase activities, apparently composed of at least one phospholipase D and two different phospholipase C activities. At low agonist concentrations, diacylglycerol and phosphatidic acid are the preferred products, and interestingly, it seems that even the primarily activated phospholipase C mainly works to increase diacylglycerol and not inositol-1,4,5-trisphosphate.

Background and purpose:We have previously shown that lipid mediators, produced by phospholipase D and C, are generated in OX1 orexin receptor signalling with high potency, and presumably mediate some of the physiological responses to orexin. In this study, we investigated whether the ubiquitous phospholipase A2 (PLA2) signalling system is also involved in orexin receptor signalling. Experimental approach: Recombinant Chinese hamster ovary-K1 cells, expressing human OX1 receptors, were used as a model system. Arachidonic acid (AA) release was measured from 3 H-AA-labelled cells. Ca 2+ signalling was assessed using single-cell imaging. Key results: Orexins strongly stimulated [ 3 H]-AA release (maximally 4.4-fold). Orexin-A was somewhat more potent than orexin-B (pEC50 = 8.90 and 8.38 respectively). The concentration-response curves appeared biphasic. The release was fully inhibited by the potent cPLA2 and iPLA2 inhibitor, methyl arachidonyl fluorophosphonate, whereas the iPLA2 inhibitors, R-and S-bromoenol lactone, caused only a partial inhibition. The response was also fully dependent on Ca 2+ influx, and the inhibitor studies suggested involvement of the receptor-operated influx pathway. The receptor-operated pathway, on the other hand, was partially dependent on PLA2 activity. The extracellular signal-regulated kinase, but not protein kinase C, were involved in the PLA2 activation at low orexin concentrations. Conclusions and implications: Activation of OX1 orexin receptors induced a strong, high-potency AA release, possibly via multiple PLA2 species, and this response may be important for the receptor-operated Ca 2+ influx. The response coincided with other high-potency lipid messenger responses, and may interact with these signals.

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